Method of using a biosensor

ABSTRACT

A biosensor for determining the concentration of an analyte in a biological sample. The biosensor comprises a support, a reference electrode or a counter electrode or both disposed on the support, a working electrode disposed on the support, the working electrode spaced apart from the other electrode or electrodes on the support, a covering layer defining a sample chamber over the electrodes, an aperture in the covering layer for receiving a sample, and at least one layer of mesh in the sample chamber between the covering layer and the electrodes. The at least one layer of mesh has coated thereon a silicone surfactant. Certain silicone surfactants are as effective as fluorinated surfactants with respect to performance of biosensors. These surfactants, when coated onto the mesh layer of the biosensor, are effective in facilitating the transport of aqueous test samples, such as blood, in the sample chamber.

REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. patentapplication Ser. No. 11/839,037, filed on Aug. 15, 2007, now U.S. Pat.No. 7,754,059, which is a continuation of U.S. patent application Ser.No. 10/448,643, filed on May 30, 2003, now U.S. Pat. No. 7,311,812,which are incorporated herein by reference in their entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to biosensors. More particularly, this inventionrelates to biosensors in which the biological sample is transported to asample chamber by means of wicking of fluid.

2. Discussion of the Art

A biosensor is a device for measuring the concentration of an analyte ina biological sample. A typical biosensor comprises a support, areference electrode or a counter electrode or both a reference and acounter electrode disposed on the support, a working electrode disposedon the support, the working electrode spaced apart from the otherelectrode or electrodes on the support, a covering layer defining anenclosed space over the electrodes, an aperture in the covering layerfor receiving a sample, and at least one mesh layer in the enclosedspace between the covering layer and the electrodes. The workingelectrode includes an enzyme capable of catalyzing a reaction involvinga substrate for the enzyme and a mediator capable of transferringelectrons between the enzyme-catalyzed reaction and the workingelectrode to create a current related to the activity of the enzyme andrelated to the concentration of the analyte in the sample.Alternatively, instead of an enzyme, the working electrode can include asubstrate capable of catalyzing a reaction involving an enzyme for thesubstrate and a mediator capable of transferring electrons between thesubstrate-catalyzed reaction and the working electrode to create acurrent related to the activity of the substrate and related to theconcentration of the analyte in the sample. The purpose of the meshlayer or mesh layers is to define a path for directional flow of thesample from the aperture through the enclosed space towards theelectrodes and control the height of the enclosed space above theelectrodes. The mesh layers are formed of a woven material and coatedwith a surfactant. An example of a biosensor is shown in U.S. Pat. No.5,628,890, incorporated herein by reference.

The test sample is required to be delivered rapidly and uniformly from asample application zone, i.e., at the aperture, to a reaction zonewithin the enclosed space, which is referred to herein as a samplechamber. Typically, delivery of the test sample is carried out bywicking along the mesh layer, which is typically of a hydrophiliccharacter for biological samples. See U.S. Pat. No. 5,628,890, EP0170375, U.S. Pat. No. 5,141,868, and U.S. Pat. No. 6,436,256. Samplechambers of biosensors are preferably constructed so that they have asmall volume for the purpose of reducing the amount of test sample(generally blood) required from a patient.

This approach has advantages in that the use of a mesh layer allows onedimension of the sample chamber to be tightly controlled while alsoreducing the void volume, thereby reducing the volume of the test samplerequired. Woven mesh layers are generally fabricated from syntheticpolymeric fibers of known diameter, typically nylon and polyesterfibers. Nylon and polyester fibers are relatively hydrophobic and,consequently, meshes constructed from the untreated fibers areunsuitable for direct use for promoting transportation of the testsample in a biosensor.

U.S. Pat. No. 5,628,890 discloses the use of a surfactant-coated meshlayer in a biosensor for the purpose of wicking A fluorinatedsurfactant, “FLUORAD FC-170C” (3M Company, St. Paul, Minn.) is disclosedas a preferred surfactant in this system. Manufacture of the fluorinatedsurfactant FC-170C was terminated by the 3M Company because of concernsrelating to its effect on the environment. Furthermore, theEnvironmental Protection Agency (EPA) has recently imposed restrictionson the manufacture and use of such surfactants and related substances inthe United States. Similar fluorinated surfactants are still availablefrom other manufacturers, but there is a legitimate concern that suchmaterials may be withdrawn from the market in the future.

Accordingly, the surfactant “FLUORAD FC-170” needs to be replaced by anequally effective non-fluorinated surfactant, preferably one that iscommercially available. A surfactant must fulfill the followingrequirements: long-term stability, ease of applying onto the mesh layer,in particular, applying by means of an aqueous solution. The GowerHandbook of Industrial Surfactants lists over 21,000 products.

Textile spin finishes are non-permanent coatings applied to fibers andyarns as emulsions in order to improve lubrication and preventantistatic build-up during processing (Philip E. Slade, Handbook ofFiber Finish Technology, Marcel Dekker (1998)). Spreading of the spinfinish emulsion on the surface of the fiber to achieve a uniform coatingis promoted by the addition of surfactants to the formulation. This typeof spreading is somewhat analogous to the situation with respect tobiosensors, where a test sample of high surface tension, i.e., blood, isapplied to a surfactant-coated mesh, where initial wetting occursfollowed by subsequent spreading. However, an important difference isthat the spin finish emulsion contains the surfactant and is applied tothe untreated fiber whereas in the biosensor, the fiber is alreadycoated with surfactant and a test sample (without surfactant) is appliedto the coated fiber.

Silicone surfactants are available from a number of manufacturers, suchas, for example, Dow Corning, OSi Specialities, Basildon Chemicals,Clariant, and Degussa. These surfactants are often used as additives(minor components) of fiber finishes, which are required duringprocessing. They are added to finish formulations to promote wetting ofthe fiber with the hydrophobic finish and are not used to increase thehydrophilicity of the finished fiber. The fiber finish is required forlubrication and anti-static properties during processing. The prior artoffers no specific guidance as to which surfactants will be effective asspreading agents when applied to mesh in biosensors.

The mechanics of the spreading/wicking process is complex. The coatingemulsion requires a low surface tension to wet the surface of the fiberor yarn, but the wicking rate is greater at a high level of surfacetension (Philip E. Slade, Handbook of Fiber Finish Technology, MarcelDekker (1998), pg. 45-48). For example, fluorinated surfactants areknown to be among the most effective at lowering surface tension but arereported to have “a considerable negative effect on wicking” (Wicking ofSpin Finishes and Related Liquids into Continuous Filament Yarns, Y. K.Kamath, S. B. Hamby, H.-D. Weigmann and M. F. Wilde, Textile Res. J.,1994, 64, 33-40). This finding is confirmed by spreading studies ofsurfactant solutions on Parafilm (K. P. Ananthapadmanabhan, E. D.Goddard and P. Chandar, Colloids Surf, 1990, 44, 281).

As stated previously, an important property of a biosensor is itslong-term stability. The biosensor is required to function without anydeterioration in performance for many months after manufacturing.Satisfactory performance requires the sample chamber to fill rapidly anduniformly over the shelf life of the product. Given that the coating ofsurfactant on the mesh layer is non-permanent and is necessary foradequate filling of the sample chamber, it follows that the surfactantitself must be chemically stable, while not undergoing excessivemigration/diffusion from the mesh layer to other surfaces in thebiosensor. Some loss of surfactant from the mesh layer to otherhydrophobic surfaces (such as printed electrode tracks) is consideredbeneficial, because these surfaces will become more hydrophilic.Excessive loss will result in an unacceptable deterioration in wickingperformance, leading ultimately to a catastrophic failure to fill.Surfactants having high molecular weight, which are either solids orviscous liquids, are expected to be less mobile and therefore morecapable of providing durable spreading capability. However, suchmaterials are expected to be less effective as spreading agents thanthose surfactants having lower molecular weights.

It is important to consider the interaction of the surfactant coatedonto the mesh layer with adjacent layers in the biosensor. Thesurfactant may inhibit adhesion of other layers to the mesh layer. Inaddition, the mesh layer may be adhered to the electrode substrate by ascreen-printed insulating ink, with which the surfactant could interactadversely. For example, the wet ink printed onto the surfactant-coatedmesh layer may wick along the fibers, resulting in poor printdefinition.

It is not a simple case of applying any surfactant (or even specificallythe most effective surfactants such as fluorinated surfactants) to amesh layer of a biosensor to achieve rapid and uniform wicking of theapplied test sample. Adequate models of the mechanics and dynamics ofthe spreading of surfactant solutions remain to be developed, largelybecause the phenomenon is so complex (Silicone Surfactants, SurfactantScience Series, Vol. 86, ed. Randall M. Hill, Marcel Dekker, 1999, pg.303-310). Furthermore, there are other critical factors to consider whenselecting a surfactant for specific use in a biosensor; many of thesefactors have not been considered previously in the literature.

In summary, a number of conflicting factors have to be balanced toobtain the optimal selection from an enormous range of commerciallyavailable surfactants. These factors include the ability to lowersurface tension, coating stability, coating uniformity, stability of thesurfactant, migration effects, adhesion inhibition effects, wickingspeed, wicking uniformity, toxicity, and printing definition.

SUMMARY OF THE INVENTION

This invention provides a biosensor for determining the concentration ofan analyte in a biological sample. The biosensor comprises a support, anarrangement of electrodes disposed on the support, a covering layerdefining an enclosed space over the electrodes, an aperture in thecovering layer for receiving a biological sample, and at least one meshlayer in the enclosed space between the covering layer and theelectrodes, the at least one mesh layer coated with at least onesilicone surfactant. The arrangement of electrodes preferably comprisesa reference electrode or a counter electrode or both a reference and acounter electrode disposed on the support, and a working electrodedisposed on the support, the working electrode spaced apart from theother electrode or electrodes on the support. The working electrodeincludes an enzyme capable of catalyzing a reaction involving asubstrate for the enzyme and a mediator capable of transferringelectrons between the enzyme-catalyzed reaction and the workingelectrode to create a current related to the activity of the enzyme andrelated to the concentration of the analyte in the sample.Alternatively, instead of an enzyme, the working electrode can include asubstrate capable of catalyzing a reaction involving an enzyme for thesubstrate and a mediator capable of transferring electrons between thesubstrate-catalyzed reaction and the working electrode to create acurrent related to the activity of the substrate and related to theconcentration of the analyte in the sample. The at least one layer ofmesh has coated thereon a silicone surfactant. We have discovered thatcertain silicone surfactants are as effective as fluorinated surfactantswith respect to performance of biosensors. These surfactants, whencoated onto the mesh layer of the biosensor, are effective infacilitating the transport of aqueous test samples, such as blood, fromthe sample application zone to the reaction zone in the enclosed space,which is frequently referred to as a sample chamber. These surfactantsare collectively referred to as silicone surfactants or siloxanesurfactants. These surfactants are preferably non-ionic and may becoated onto a layer of polymeric mesh, such as, for example, nylon orpolyester mesh.

The silicone surfactants combine a number of properties that arerequired for successful use in a biosensor. The overall performance ofthe silicone surfactants in the biosensor exceeds that of fluorinatedsurfactants, such as “FLUORAD FC-170C.” Overall performance is based onthe following parameters:

-   -   (a) speed of filling the sample chamber with the sample;    -   (b) uniformity of filling the sample chamber with the sample,        i.e., straightness of filling front for the sample;    -   (c) shelf life (filling stability), preferably at least 18        months;    -   (d) minimization of adhesion failure between layers of the        biosensor in contact with the surfactant;    -   (e) minimization of seepage of sample between layers of the        biosensor;    -   (f) level of toxicity, i.e., non-toxicity being preferred;    -   (g) minimization of loss in printing definition of the ink layer        that holds the mesh layer in place;    -   (h) transferability of liquid surfactant by contact to other        surfaces in the biosensor to render them more hydrophilic.

Silicone surfactants are effective at reducing the surface tension ofaqueous fluids, such as blood. Consequently, hydrophobic meshes coatedwith silicone surfactants are capable of being wetted by aqueous fluids,such as blood.

Rapid and uniform wicking of blood along the at least one mesh layer ofa biosensor is desired for reproducible results. The time required tofill a sample chamber of a biosensor containing a mesh layer coated witha silicone surfactant exceeds that of a sample chamber of a biosensorcontaining a mesh layer coated with a fluorinated surfactant, such as“FLUORAD FC-170C.” Wicking uniformity (straightness of moving liquidfront) across mesh coated with silicone surfactants is superior to thatacross a mesh coated with a fluorinated surfactant, such as “FLUORADFC-170C.” Wicking/spreading rates vary according to the structure of thesilicone surfactant. Silicone surfactants having low molecular weightare very efficient spreading agents, but lack the durability requiredfor a biosensor. Durability must be balanced with spreading efficiency.For this reason, mixtures of silicone surfactants having differentproperties provide the best overall performance in a biosensor.

Biosensors having sample chambers containing at least one mesh layercoated with silicone surfactants have adequate long-term stability. Thesample chambers continue to fill rapidly and uniformly for at least 18months when stored at 30° C. A shelf life of longer than 18 months isnot required for a biosensor. Long-term stability is desired so that acatastrophic failure is not observed near the end of shelf life.

Silicone surfactants are non-toxic and do not irritate the skin. Incontrast, fluorinated surfactants are toxic. Silicone surfactants arefreely available from a number of suppliers.

In some biosensor systems the surfactant-coated mesh layer is coveredwith a polymeric film to form the sample chamber. Good adhesion betweenthe mesh layer/insulating layer and the polymeric film is important toensure that the sample chamber remains intact and to specify the volumeof the test sample required to fill the sample chamber. If adhesion werepoor, the polymeric film could peel away or seepage of the test samplemay occur between the polymeric film and the mesh layer/insulating layerat the edge of the sample chamber. Such seepage will increase the volumeof sample required to fill the sample chamber. Silicone surfactants haveno adverse effect on the adhesion between the layers forming the samplechamber.

In some biosensors, the surfactant-coated mesh layer is held in place byoverprinting with a layer of insulating ink. The surfactant coating maypromote wicking of the wet insulating ink along the fibers of the meshlayer, leading to a poor print definition. In extreme cases, theinsulating ink could cover areas that are required to be exposed.Silicone surfactants are comparable to fluorinated surfactants such as“FLUORAD FC-170C” in providing satisfactory definition of the insulatingink layer in the biosensor, when applied to the mesh layer at anequivalent level.

The biosensors of this invention employ non-toxic and environmentallyfriendly silicone surfactants in place of fluorinated surfactants (e.g.,“FLUORAD FC-170.” The silicone surfactants act as wetting agents whenapplied to polymeric meshes, such as polyamide (e.g., nylon) andpolyester (e.g., PET). The hydrophobic polyester and polyamide meshes,when coated with silicone surfactants, become hydrophilic, and hencepromote the lateral transport/flow/wicking of an aqueous sample, such asblood, from a sample application zone to a reaction zone in a diagnosticassay device, such as a biosensor.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an exploded perspective view of a biosensor suitable for usein this invention.

FIG. 2 is a series of video images (individual frames) showing waterwicking into sample chambers of a model biosensor.

FIG. 3 is a series of video images (individual frames) showing bloodwicking into sample chambers of a model biosensor.

FIG. 4 is a series of video images (individual frames) showing bloodwicking into sample chambers of a manufactured biosensor.

FIG. 5 is a graph showing coated weight of surfactant as a function ofconcentration of surfactant in coating solution.

FIG. 6 is a graph showing coated weight of surfactant as a function ofconcentration of surfactant in coating solution.

FIG. 7 is a graph showing coated weight of surfactant as a function ofconcentration of surfactant in coating solution.

FIG. 8A is a graph showing percentage of biosensors that would fill as afunction of storage time and storage temperature.

FIG. 8B is a graph showing percentage of biosensors that would fill as afunction of storage time and storage temperature.

FIG. 9 is a graph showing shelf life of biosensors at a given storagetemperature as a function of concentration of surfactant in coatingbath.

FIG. 10A is a graph showing percentage of sample chambers of biosensorsthat would fill as a function of storage time and storage temperature.

FIG. 10B is a graph showing the time to fill the sample chambers ofbiosensors as a function of storage time and storage temperature.

FIG. 11A is a graph showing percentage of sample chambers of biosensorsthat would fill as a function of storage time and storage temperature.

FIG. 11B is a graph showing time to fill the sample chambers ofbiosensors as a function of storage time and storage temperature.

FIG. 12 is a contour plot showing variation in time of filling of abiosensor having a single mesh layer as a function of both the type ofsurfactant used and the storage time, where storage temperature was 50°C.

FIG. 13A are video images showing sample chambers of biosensors havingtwo mesh layers filled with blood. The biosensors were stored at ambienttemperature and the surfactants were DC 193 and FSN-100.

FIG. 13B are video images showing sample chambers of biosensors havingtwo mesh layers filled with blood. The biosensors were stored at 40° C.for four weeks and the surfactants were DC 193 and FSN-100.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the terms “silicone” and “siloxane” are synonymous. Theterm “silicone” denotes a synthetic polymer (R_(n)SiO_((4-n)/2))_(m),where n=1 to 3, inclusive and m is equal to or greater than 2. Asilicone contains a repeating silicon-oxygen backbone and has organicgroups R attached to a significant proportion of the silicon atoms bysilicon-carbon bonds. In commercial silicones most R groups are methyl;longer alkyl, fluoroalkyl, phenyl, vinyl, and a few other groups aresubstituted for specific purposes. Some of the R groups can also behydrogen, chlorine, alkoxy, acyloxy, or alkylamino, etc. These polymerscan be combined with fillers, additives, and solvents to result inproducts classed as silicones. See Kirk-Othmer Encyclopedia of PolymerScience and Technology, Volume 15, John Wiley & Sons, Inc. (New York:1989), pages 204-209, 234-265, incorporated herein by reference.

This invention provides a biosensor for determining the concentration ofan analyte in a biological sample. The biosensor comprises a support, areference electrode or a counter electrode or both a reference and acounter electrode disposed on the support, a working electrode disposedon the support, the working electrode spaced apart from the otherelectrode or electrodes on the support, a covering layer defining asample chamber over the electrodes, an aperture in the covering layerfor receiving a sample, and at least one layer of mesh in the samplechamber between the covering layer and the electrodes on the support.The working electrode includes an enzyme capable of catalyzing areaction involving a substrate for the enzyme and a mediator capable oftransferring electrons between the enzyme-catalyzed reaction and theworking electrode to create a current related to the activity of theenzyme and related to the concentration of the analyte in the sample.Alternatively, instead of an enzyme, the working electrode can include asubstrate capable of catalyzing a reaction involving an enzyme for thesubstrate and a mediator capable of transferring electrons between thesubstrate-catalyzed reaction and the working electrode to create acurrent related to the activity of the substrate and related to theconcentration of the analyte in the sample. The at least one layer ofmesh has coated thereon at is least one silicone surfactant.

By applying certain silicone surfactants to the at least one layer ofmesh, which is typically constructed of a hydrophobic polymericmaterial, the at least one layer of mesh is rendered hydrophilic and canbe wetted by water and water-borne solutions such as blood. This treatedmesh facilitates the transport/flow/wicking of aqueous reagents, such asblood, to a reaction zone covered by the at least one layer of mesh,where the active ingredients of the biosensor are located for aquantitative assay. Without aid of the at least one surfactant, it wouldbe impossible to wet the reaction zone by the reagents alone.

Representative examples of classes of silicone surfactants suitable foruse in this invention are illustrated below:

Formula I: molecular structure of a rake-type silicone surfactant; alsocalled a comb or graft copolymer; R represents an end-capping group suchas —H, —CH₃, or —COCH₃.

Formula II: molecular structure of an ABA-type silicone surfactant; alsocalled α-ω, or bolaform surfactant

Formula III: molecular structure of a trisiloxane surfactant

Formula IV: molecular structure of a cyclosiloxane surfactant

The silicone surfactants that are preferred for use in this inventionhave the general formula:

wherein:

-   -   z represents a value of 3 to 25, inclusive, preferably 3 to 15;    -   R represents hydrogen, an alkyl group, preferably having 1 to 4        carbon atoms, or an alkyl ester group, preferably having 1 to 4        carbons atoms in the alkyl portion, where, if y>1, each R can be        the same or different on any given molecule, with R preferably        being hydrogen or methyl;    -   x represents 0 up to a value in combination with y and z where        the surfactant is not a liquid, x preferably being zero for the        trisiloxane surfactants useful in surfactant mixtures;    -   y represents 1 up to a value in combination with x and z where        the surfactant is not a liquid.

In general, non-ionic silicone surfactants comprise two structuralunits, a silicone group and a polyalkylene oxide chain; non-ionicsilicones are preferred over cationic, anionic, or amphoteric silicones,because non-ionic surfactants allow the covering tape to readily adhereto the layer of coated mesh. The polyalkylene oxide chain preferablycomprises ethylene oxide (EO) units or propylene oxide (PO) units or amixture of the two. Varying the polyalkylene chain length and EO/POratio varies the properties of the surfactants. For example, increasedwater solubility is seen for those surfactants having a high ratio of EOto PO. In addition, surfactants having a short polyalkylene chain areknown to be “superwetting agents.” Furthermore, surfactants having highmolecular weight are viscous liquids, which have good stability whencoated onto polymeric fibers, due to a low mobility.

Surfactants that are particularly suitable for this invention have thefollowing characteristics:

-   -   (a) Surface tension range: 20 to 35 mN/m    -   (b) Estimated viscosity range: 1 to 5000 cSt    -   (c) Estimated specific gravity range: 0.98 to 1.20    -   (d) Solubility or dispersibilty in water and alcohols: up to        10%.    -   (e) Cloud point: preferably >25° C. at 10% concentration in        coating solution of choice (water/alcohol mixture)    -   (f) Molecular weight: number average molecular weight based on        number of surfactants ranges from about 500 to about 30,000    -   (g) Additives: no additives are deliberately added beyond the        manufacturer's formulation, which may contain additives or        stabilizers or both    -   (h) Surfactant should be a liquid coating that can transfer to        other surfaces by contact

The silicone surfactant preferred for this invention has the trade nameDow Corning 193 Fluid, because it has a good balance of aqueoussolubility, wetting ability, and stability. This silicone surfactant hasa preferred rake-type structure (see the structures shown in Formulas Iand V) and contains only EO with no PO. Dow Corning 193 Fluid has amolecular weight of approximately 2500.

The choice of a single surfactant for coating fibers of a mesh layerrequires a compromise between various conflicting factors. However, amixture of surfactants applied to the fibers of a mesh layer may achievean enhanced effect, because a combination of “superwetting” agent andhigh molecular weight products can be used. A “superwetting” agent alonewill have poor stability, and a surfactant having very high molecularweight will have poor wetting properties and aqueous solubility, butgood stability.

The surfactant or mixture of surfactants is first dissolved in anorganic is solvent (e.g., acetone), water, or a mixture of water andorganic solvent (e.g., water/isopropanol) to yield a solution having aconcentration in the range 0.01 to 10%, based on weight. The polymericmeshes are typically in a roll format and have various dimensions.Coating of the surfactant is achieved by continuously transporting thepolymeric mesh from one end to the other through a bath of the solutionof surfactant at a constant speed. A drying process is used to removethe solvent from the coating composition. In the drying process,temperatures of up to 130° C. can be used.

Silicone surfactants are environmentally friendly and non-toxic, ascompared with fluorinated surfactants, such as “FLUORAD FC-170C”, whichare toxic and persistent in the environment. Silicone surfactants are atleast equivalent to the fluorinated surfactants currently available,with respect to performance of current biosensor strips. The samplechambers fill rapidly and uniformly with blood over the shelf life ofthe strips. Silicone surfactants are manufactured by many companies;hence, there is no problem of shortage of suppliers, in contrast tofluorinated surfactants.

A stable, rapidly-fillable sample chamber containing a surfactant-coatedmesh system for a biosensor can be obtained where through the use of asingle liquid silicone surfactant of Formula V, where x ranges from 7 to12, inclusive, y ranges from 3 to 5, inclusive, z ranges from 3 to 15,inclusive, R being hydrogen or methyl. Preferably, x ranges from 8 to 9,inclusive, y ranges from 3 to 4, inclusive, z ranges from 11 to 13,inclusive, R being hydrogen. A silicone surfactant having these valuesof x, y, and z is similar to Dow Corning 193 Fluid. Commercialsurfactants typically have a range of x, y, and z values, whereby adistribution of molecular weights is obtained.

A stable, rapidly fillable sample chamber containing a surfactant-coatedmesh system for a biosensor can be obtained through the use of a mixtureof two silicone surfactants. Preferably, the mixture comprises asurfactant having high molecular weight having satisfactory propertieswith respect to stability of the biosensor and a surfactant having lowmolecular weight having satisfactory properties for rapid filling of thesample chamber. Most preferably, in the surfactant of high molecularweight (Formula V), x ranges from 8 to 9, inclusive, y ranges from 3 to4, inclusive, z ranges from 11 to 13, inclusive, R being H, i.e.,similar to Dow Corning 193 Fluid. Most preferably, in the surfactanthaving low molecular weight (Formula V), preferably a trisiloxanesurfactant, x is 0, y is 1, z ranges from 3 to 15, inclusive, R ishydrogen, methyl, or acetate.

The weight fraction of the silicone surfactant having high molecularweight in the mixture preferably ranges from 1% to 99%, inclusive. Theweight fraction of the silicone surfactant having low molecular weightin the mixture preferably ranges from 1% to 99%, inclusive. The mostpreferred weight fraction for the silicone surfactant having highmolecular weight ranges from 50% to 99%, inclusive, and the mostpreferred weight fraction for the silicone surfactant having lowmolecular weight ranges from 1% to 50%, inclusive.

The coating weight of the surfactant on the layer of mesh preferablyranges from about 0.01% to about 8%, based on the weight of the mesh.

Preferably, a mixture of surfactants suitable for the present inventionwill have two components as described previously. However, mixtures ofsurfactants containing three or more silicone surfactants can be used toachieve optimal effects.

A biosensor strip 10 suitable for this invention is illustrated inFIG. 1. Referring to FIG. 1, an electrode support 11, preferably anelongated strip of polymeric material (e.g., polyvinyl chloride,polycarbonate, polyester, or the like) supports three tracks 12 a, 12 b,and 12 c of electrically conductive ink, preferably comprising carbon.These tracks 12 a, 12 b, and 12 c determine the positions of electricalcontacts 14 a, 14 b, and 14 c, a reference electrode 16, a workingelectrode 18, and a counter electrode 20. The electrical contacts 14 a,14 b, and 14 c are insertable into an appropriate measurement device(not shown). This type of biosensor is shown in U.S. Ser. No.10/062,313, filed Feb. 1, 2002, now U.S. Pat. No. 6,863,800,incorporated herein by reference. While this illustration involves abiosensor having a working electrode, a reference electrode, and acounter electrode, it is not critical for a biosensor to have threeelectrodes. One electrode can be used to perform the functions of thereference electrode and the counter electrode. Auxiliary electrodes canbe added for other purposes. What is required is an electrodearrangement comprising at least a working electrode and a referenceelectrode. A type of biosensor having a working electrode and a singleelectrode to perform the functions of the reference electrode and thecounter electrode is shown in WO 99/19507, published 22 Apr. 1999,incorporated herein by reference.

Referring again to FIG. 1, each of the elongated portions of theconductive tracks 12 a, 12 b, and 12 c can optionally be overlaid with atrack 22 a, 22 b, and 22 c of conductive material, preferably made of amixture comprising silver particles and silver chloride particles. Theenlarged exposed area of track 22 b overlies the reference electrode 16.A layer of a hydrophobic electrically insulating material 24 furtheroverlies the tracks 22 a, 22 b, and 22 c. The positions of the referenceelectrode 16, the working electrode 18, the counter electrode 20, andthe electrical contacts 14 a, 14 b, and 14 c are not covered by thelayer of hydrophobic electrically insulating material 24. Thishydrophobic electrically insulating material 24 serves to prevent shortcircuits. The layer of hydrophobic electrically insulating material 24has an opening 26 formed therein. This opening 26 provides the boundaryfor the reaction zone of the biosensor strip 10. Because this insulatingmaterial is hydrophobic, it can cause the sample to be restricted to theportions of the electrodes in the reaction zone. The working electrode18 comprises a layer of a non-reactive electrically conductive materialon which is deposited a layer 28 containing a working ink for carryingout an oxidation-reduction reaction. At least one layer of mesh 30overlies the electrodes. This mesh layer 30 protects the printedcomponents from physical damage. The mesh layer 30 also helps the sampleto wet the electrodes by reducing the surface tension of the sample,thereby allowing it to spread evenly over the electrodes. A cover 32encloses the surfaces of the electrodes that are not in contact with theelectrode support 11. This cover 32 is a liquid impermeable membrane.The cover 32 includes a small aperture 34 to allow access of the appliedsample to the underlying mesh layer 30.

The layer of working ink 28 is deposited on that portion of theelectrically conductive material of the working electrode 18 where theoxidation-reduction reaction is to take place when a sample isintroduced to the biosensor strip 10. The layer of the working ink 28can be applied to the working electrode 18 as a discrete area having afixed length. The working ink comprises reagent(s) that are responsiveto the analyte of interest deposited on the non-reactive electricallyconductive material. As used herein, the term “reagent(s)” means atleast one reagent. Typical analytes of interest include, for example,glucose and ketone bodies. Typical non-reactive electrically conductivematerials include, for example, carbon, platinum, palladium, and gold. Asemiconducting material such as indium doped tin oxide can be used asthe non-reactive electrically conductive material. In preferredembodiments, the working ink comprises a mixture of anoxidation-reduction (redox) mediator and an enzyme. Alternatively,instead of an enzyme, the working ink can contain a substrate that iscatalytically reactive with an enzyme to be assayed. For example, whenthe analyte to be measured is glucose in blood, the enzyme is preferablyglucose oxidase, and the redox mediator is preferably ferrocene or aderivative thereof. Other mediators that are suitable for use in thisinvention include a ferricyanide salt and a phenanthroline quinone or aderivative thereof. In the biosensor strips of this invention, thereagent(s) are preferably applied in the form of ink containingparticulate material and having binder(s), and, accordingly, does notdissolve rapidly when subjected to the sample. In view of this feature,the oxidation-reduction reaction will occur at the interface of workingelectrode 18 and the sample. The glucose molecules diffuse to thesurface of the working electrode 18 and react with the enzyme/mediatormixture.

In addition to being applied to the working electrode 18, a layer of theworking ink can be applied to any of the other electrodes, when desired,as a discrete area having a fixed length.

The thickness of the layer of non-reactive electrically conductivematerial is determined by the method of applying the layer. In the caseof a layer deposited by printing, e.g., screen-printing, the thicknessof the layer typically ranges from about 10 micrometers to about 25micrometers. In the case of a layer deposited by vapor deposition, thethickness of the layer typically ranges from about less than 1micrometer to about 2 micrometers. The layer of the working ink 28 thathas been deposited on the working electrode 18 typically has a drythickness of from about 2 to about 50 micrometers, preferably from about10 to about 25 micrometers. The actual dry thickness of the depositedlayer of the working ink 28 will depend to some extent upon thetechnique used to apply the working ink. For example, a thickness offrom about 10 to about 25 micrometers is typical for a layer of workingink applied by means of screen-printing.

The reference electrode 16 is typically formed by screen-printing amixture comprising a mixture of silver and silver chloride on theelectrode substrate 11. For materials to which such a mixture does notreadily adhere, it is preferred to deposit a layer of carbon on theelectrode support to act as a primer layer for the mixture. This mixtureis often referred to as ink. The mixture typically has a carriercomprising an organic solvent. Alternatives to the mixture of silver andsilver chloride include mixtures of Ag and AgBr, mixtures of Ag and Ag1,and mixtures of Ag and Ag₂O. The printed layer associated with thereference electrode 16 extends to partially cover the track of thecarbon layer associated with the reference electrode 16, where theprinted layer extends into the reaction zone. It is preferred to coverparts of the tracks 12 a, 12 b, and 12 c outside the reaction zone withthe mixture of silver and the silver compound associated therewith, sothat the total electrical resistance of each track is reduced. Becauseno current flows through the reference electrode 16, non-classicalreference electrodes can be used as the reference electrode. Thesenon-classical electrodes can be formed either by simply employing aconductive material, such as, for example, carbon, platinum, orpalladium, as the reference electrode or by having the working inkdeposited on the conductive material that forms the reference electrode.The reference electrode 16 preferably has equal or smaller dimensionscompared to those of the working electrode 18.

If the working ink is deposited on a conductive material to form thereference electrode 16, the reagent(s) are deposited only on the portionof the electrode that is in the reaction zone to minimize the electricalresistance of the track 12 c.

In the case of carbon being deposited to form the reference electrode 16(i.e., an electrically conductive electrode without oxidation-reductionreagents), no additional material is required to be deposited on thesurface of the reference electrode. The carbon can be doped with metalparticles to increase the conductivity of the carbon.

Any electrically conductive material can be used to form the counterelectrode 20. Preferred materials for forming the counter electrode 20include, but are not limited to, platinum, palladium, carbon, gold,silver, and mixtures of silver and silver chloride (as in the referenceelectrode 16). In another embodiment, reagent(s) that form the workingink can be deposited on the conductive material of the counter electrode20. If the working ink is deposited on a conductive material to form thecounter electrode 20, the reagent(s) are deposited only on the portionof the electrode that is in the reaction zone to minimize the electricalresistance of the track 12 b.

The dimensions of the counter electrode 20 are preferably equal to orgreater than those of the reference electrode 16. It is preferred thatthe counter electrode 20 be of a size equal to or greater than theworking electrode 18, though this preference is not required at lowlevels of current. In functional terms, the size of the referenceelectrode is not critical; the size of the working electrode is selectedon the basis of signal to noise ratio desired; the size of the counterelectrode is selected to minimize resistance to current flow.

The counter electrode 20 must be in electrical contact with the workingelectrode 18 during the measurement. When current flows through thecounter electrode 20, the flow of electrons produces an electrochemicalreaction (a reduction reaction) sufficient to allow the electrons toflow. The counter electrode 20 must be positioned at a sufficientdistance from the working electrode 18 so that the reactive speciesgenerated at the counter electrode 20 do not diffuse to the workingelectrode 18.

The reaction zone can have total area ranging from about 1 mm² to about20 mm², preferably about 5 mm². The area of the working electrodetypically ranges from about 0.5 to about 5 mm², preferably about 1.0mm². The reference electrode and the counter electrode typically haveareas ranging from about 0.2 to about 4.0 mm², preferably about 0.5 mm².

The biosensor strip 10 typically has a width of from about 4.5 to about6.5 mm. The electrode support 11 can be made from any material that hasan electrically insulating surface, such as, for example, polyvinylchloride, polycarbonate, polyester, paper, cardboard, ceramic,ceramic-coated metal, and blends of these materials (e.g., a blend ofpolycarbonate and polyester).

Electrically conductive material can be applied to the electrode support11 by a deposition method such as screen-printing. This deposit ofelectrically conductive material forms the contact areas 14 a, 14 b, and14 c, which areas allow the analyte monitor to interface with thebiosensor strip 10. The conductive material further provides electricalconnections between the contact areas and the active reagent(s)deposited on the electrode(s) of the biosensor strip 10. The formulationfor the electrically conductive material can be an air-dryablecomposition comprising carbon dispersed in an organic solvent.Alternative formulations include carbon dispersed in an aqueous solvent.Alternative electrically conductive materials that can be used in placeof carbon include, but are not limited to, such materials as silver,gold, platinum, and palladium. Other methods of drying or curing theformulations containing the electrically conductive material include theuse of infrared radiation, ultraviolet radiation, and radio frequencyradiation. In an alternative method of application, the electricallyconductive material can be deposited by means of a vapor depositiontechnique.

As stated previously, inks suitable for use in this invention can bescreen-printed. Other ways of depositing the inks include drop coating,inkjet printing, volumetric dosing, gravure printing, flexographicprinting, and letterpress printing. The electrically conductive portionsof the electrodes are preferably screen-printed or deposited by means ofsputtering or vapor deposition techniques. The reagents are preferablydeposited by screen-printing or drop coating the formulations on thesurface of the electrically conductive portion of the electrode. In thecase of screen-printing, the reagents can be converted into particulateform wherein the particles contain carbon or silica, with carbon beingpreferred. In the drop coating formulation, the reagents can be mixedwith a polymer (such as, for example, carboxy methyl cellulose, hydroxyethyl cellulose, polyvinyl alcohol, etc.) solution to obtain a viscoussolution, which is then dispensed on the area of interest. The inks canfurther include a polysaccharide (e.g., a guar gum, an alginate, locustbean gum, carrageenan, or xanthan), a hydrolyzed gelatin, an enzymestabilizer (e.g., glutamate or trehalose), a film-forming polymer (e.g.,a polyvinyl alcohol, hydroxyethyl cellulose, polyvinyl pyrrole,cellulose acetate, carboxymethyl cellulose, and poly(vinyloxazolidinone), a conductive filler (e.g., carbon), a defoaming agent, abuffer, or combinations of the foregoing. Other fillers for the inksinclude, but are not limited to, titanium dioxide, silica, and alumina.

It is preferred that the length of the path to be traversed by thesample (i.e., the reaction zone) be kept as short as possible in orderto minimize the volume of sample required. With respect to the biosensorstrip described herein, the volume of sample required is preferably nogreater than 5 microliters, and more preferably ranges from about 0.5microliters to about 2.5 microliters. The maximum length of the reactionzone can be as great as the length of the biosensor strip. However, thecorresponding increase in resistance of the sample limits the length ofthe reaction zone to a distance that allows the necessary responsecurrent to be generated. Positioning the electrodes in the mannerdescribed herein has the further advantage of preventing completion of acircuit (and thus preventing detection of a response current) before theworking electrode 18 has been completely covered by the sample.

As shown in FIG. 1, a mesh layer 30 overlies the electrodes. As statedpreviously, this mesh layer 30 protects the printed components fromphysical damage, and the mesh layer 30 also helps the sample to wet theelectrodes by reducing the surface tension of the sample, therebyallowing it to spread evenly over the electrodes. Preferably, this meshlayer 30 extends over the entire length of the reaction zone, betweenand including the position at which the sample is introduced and theregion where the electrodes are disposed. Preferably, this mesh layer 30is constructed of woven strands of polyester. Alternatively, any wovenor non-woven material can be used, provided that it does not occlude thesurface of the electrode such that normal diffusion of the sample isobstructed. The thickness of the mesh is selected so that the depth ofthe sample is sufficiently low that a high sample resistance isproduced. Preferably, the mesh layer 30 is not more than 150 μm inthickness. Preferably, the mesh layer 30 has a percent open area ofabout 35% to about 45%, a fiber count of about 40 per cm to about 60 percm, a fiber diameter of about 70 μm to about 100 μm, an average meshopening of about 67 to about 180 μm, and a thickness of from about 100μm to about 160 μm. The diameter of the fiber can be outside thepreferred range, e.g., about 10 μm to about 1000 μm. A particularlypreferred mesh is PE130 HD mesh, available from Sefar (formerly ZBF),CH-8803, Ruschlikon, Switzerland.

The mesh layer 30 is coated with a surfactant. A surfactant coating isnecessary only if the material of the mesh layer 30, itself, ishydrophobic (for example, nylon or polyester). If the material of themesh layer 30 is hydrophilic, the surfactant coating can be used or canbe omitted. A surfactant loading of from about 15 to about 20 μg/mg ofmesh is preferred for most applications. The preferred surfactantloading will vary depending on the type of mesh layer and surfactantused and the sample to be analyzed. The preferred surfactant loading canbe determined empirically by observing flow of the sample through themesh layer 30 with different levels of surfactant.

The mesh layer 30 can be held in place by the layer of hydrophobicelectrically insulating material 24. This layer of electricallyinsulating material 24 is preferably applied by screen-printing the inkover a portion of the periphery of the mesh layer 30. Together, the meshlayer 30 and the layers of hydrophobic electrically insulating material24 surround and define a reaction zone 30 suitable for the sample totravel from the position at which the sample is introduced at one end ofthe strip towards the reference electrode 16, then toward the workingelectrode 18, and then toward the counter electrode 20. The hydrophobicelectrically insulating material 24 impregnates the mesh layer 30outside of the reaction zone 30. The hydrophobic electrically insulatingmaterial 24 thus defines the reaction zone 30 by preventing the samplefrom infiltrating the portions of the mesh layer 30 covered by thelayers of hydrophobic electrically insulating material 24. A hydrophobicelectrically insulating material 24 preferred for impregnating the meshlayers is “SERICARD” (Sericol, Ltd., Broadstairs, Kent, UK). Anotherpreferred hydrophobic electrically insulating material is commerciallyavailable as “POLYPLAST” (Sericol Ltd., Broadstairs, Kent, UK).

A layer of dielectric ink can optionally be applied to cover themajority of the printed carbon and silver/silver chloride tracks. Inthis case, two areas are left uncovered, namely the electrical contactareas and the sensing areas in the reaction zone. This layer ofdielectric ink serves to define the area constituting the reaction zone,and to protect exposed tracks from short circuit.

As shown in FIG. 1, a cover 32 encloses the surfaces of the electrodesthat are not in contact with the electrode support 11. The cover 32 is aliquid impermeable membrane. This cover 32 can be a flexible tape madeof polyester or similar material. The cover 32 includes a small aperture34 to allow access of the applied sample to the underlying mesh layer30. This cover 32 encloses the exposed surfaces of the working electrode18, the reference electrode 16, and the counter electrode 20. Thus, thecover 32 maintains the available sample space over the electrodes at afixed depth, which is equivalent to the thickness of the mesh layer 30.The positioning of this cover 32 ensures that the resistance of thesample is maintained at a high level.

The aperture 34 is positioned to overlie an end of the mesh areaupstream of the reference electrode 16, such that the exposed mesh areabeneath the aperture 34 can be used as a point of access or applicationfor a liquid sample, whereby the sample contacts the reference electrode16 before the sample contacts the working electrode 18 and the counterelectrode 20. Of course, the aperture 34 must overlie an end of the mesharea that is not covered by the hydrophobic electrically insulating ink30.

The size of this aperture 34 is not critical, but it should besufficiently large to allow sufficient volume of sample to pass throughto the mesh layer 30. The aperture 34 should not be so large as to allowany portion of the liquid sample to contact any of the electrodes beforecontacting the mesh layer 30. The aperture 34 can be formed in theliquid impermeable cover 32 by any suitable method (e.g., die punching).

The liquid impermeable cover membrane 32 can be affixed to the biosensorstrip by means of a suitable method of adhesion. Preferably, affixing isachieved by coating the underside of the flexible tape with a layer ofhot melt glue, and then heat welding the tape to the surface of thelayer of hydrophobic electrically insulating ink 24. The layer of hotmelt glue typically has a coating weight of from about 10 to about 50g/m², preferably from about 20 to about 30 g/m². Pressure sensitiveadhesives or other equivalent methods of adhesion may also be used. Careshould be taken when the tape is applied, because the heat and pressureapplied to the tape layer can melt the “SERICARD” ink and can cause itto smear onto adjoining areas. Care should also be taken so that thetape does not cover the electrodes, the reaction zone, or the area wherethe sample is applied.

The upper surface of the liquid impermeable cover 32 can also beprovided with a layer of silicone or other hydrophobic material. Thisadditional layer serves to drive the applied sample onto the portion ofexposed mesh layer 30 at the sample application point, thereby renderingthe application of small volumes of sample much simpler.

In use, a biosensor strip 10 of this invention is connected, viaelectrode contacts 14 a, 14 b, and 14 c, to a measuring device (notshown). A liquid sample is applied through aperture 34, and the samplemoves along the reaction zone. The progress of the sample issufficiently impeded by the mesh layer 30, thereby allowing the sampleto form a uniform flow front. Air is displaced through the upper portionof the mesh layer 30 to and through the aperture 34. The sample firstcompletely covers the working electrode 18 and the reference electrode16, and only then approaches and covers and the counter electrode 20,thereby completing the circuit and causing a response to be detected bythe measuring device.

Measuring devices that are suitable for use in this invention includeany commercially available analyte monitor that can accommodate abiosensor strip having a working electrode, a reference electrode, and acounter electrode. Such analyte monitors can be used to monitoranalytes, such as, for example, glucose and ketone bodies. In general,such a monitor must have a power source in electrical connection withthe working electrode, the reference electrode, and the counterelectrode. The monitor must be capable of supplying an electricalpotential difference between the working electrode and the referenceelectrode of a magnitude sufficient to cause the electrochemicaloxidation of the reduced mediator. The monitor must be capable ofsupplying an electrical potential difference between the referenceelectrode and the counter electrode of a magnitude sufficient tofacilitate the flow of electrons from the working electrode to thecounter electrode. In addition, the monitor must be capable of measuringthe current produced by the oxidation of the reduced mediator at theworking electrode.

In this invention, the liquid sample is preferably a sample of wholeblood. Alternatively, the liquid sample can be whole blood that has beenfiltered or treated to remove red blood cells or other hemocytes. Otherbiological samples, such as, for example, plasma, serum, urine, saliva,interstitial fluid, can be used.

The following non-limiting examples further illustrate this invention.

EXAMPLES

The surfactants used in the examples are listed in Table 1.

TABLE 1 Identifier Trade Name Manufacturer Type S-20 Span 20 ICISorbitan laurate ester G2109 Atlas G2109 ICI Lauric acid ethoxylatePP822 Cirrasol PP822 ICI Formulated surfactant mixture of mainlysorbitan ester, sorbitol ester, fatty acid ethoxylate DC 193 Dow CorningDow Corning Silicone surfactant containing ethylene 193 oxide (EO) DC190 Dow Corning Dow Corning Silicone surfactant containing ethylene 190oxide (EO) and propylene oxide (PO) FSO-100 Zonyl FSO-100 DuPontPerfluoroalkylethoxylate FSN-100 Zonyl FSN-100 DuPontPerfluoroalkylethoxylate FC-170C Fluorad FC- 3MPerfluoroalkylsulfonamido oxyethylene 170C adduct OT-100 Aerosol OT-100Cytec Sodium dioctyl sulfosuccinate BC2213 BC2213 Basildon Trisiloxanesurfactant containing Chemicals ethylene oxide (EO) BC2234 BC2234Basildon Silicone surfactant containing ethylene Chemicals oxide (EO)PP3 Duron PP3 Hansa Formulated mixture of fatty acid ester, TexilChemiefatty acid ethoxylate(s), polyglycolic ether and amphoteric antistatics

Example 1

The purpose of this example was to qualitatively assess the spreadingability of water onto a nylon mesh coated with a surfactant.

Various types of surfactants (TABLE 2) were coated onto strips of nylonmesh (15 mm wide, NY151, Sefar, Switzerland). Coating was performed byimmersing the mesh in an aqueous acetone solution containing the givensurfactant (0.1% w/w). The immersed mesh was then slowly withdrawn fromthe solution and dried in an oven at a temperature of 50° C. for twodays. Two nylon mesh control samples, one uncoated and one coated withthe surfactant FC-170C, were employed.

Short lengths of mesh coated with the various surfactants weresandwiched between a polyester sheet and a transparent polyester film bymeans of a tape having both major surfaces thereof coated with anadhesive (double-sided adhesive tape). This arrangement provided a modelsample chamber with a height of approximately 180 to 190 μm, as measuredby a micrometer.

Colored water (10 μl) was applied by automatic pipette (Gilson) to theedge of the model sample chamber. The progress of the water as it wasdrawn into the sample chamber was recorded by a high-speed video cameraat a speed of 16 frames per second. Examples of captured video imagesare shown in FIG. 2. The small number in the upper left corner of eachframe represents the number of the frame from the introduction of thesample. The surfactant used was G2109. No spreading of water in themodel sample chamber was seen for the uncoated control and the meshcoated with surfactants S-20, PP822, and FSO-100. These threesurfactants were not included in further examples. In contrast, waterdid wick into model sample chambers containing mesh coated withsurfactants FC-170C (control), G2109, PP3, DC 193, DC 190, and FSN-100.

TABLE 2 Spreading of Identifier Trade Name water seen S-20 Span 20 NoG2109 Atlas G2109 Yes PP822 Cirrasol PP822 No DC 193 Dow Corning 193 YesDC 190 Dow Corning 190 Yes FSO-100 Zonyl FSO-100 No FSN-100 ZonylFSN-100 Yes PP3 Duron PP3 Yes FC-170C Fluorad FC-170C Yes (prior artcontrol) N/A N/A No (uncoated control)

Example 2

The purpose of this example was to qualitatively assess the spreadingability of blood onto a nylon mesh coated with a surfactant.

Model sample chambers were constructed using nylon mesh from Example 1that had been coated with the surfactants that were successful inspreading water (TABLE 3). Again, two nylon mesh control samples, oneuncoated and one coated with the surfactant FC-170C, were employed.

Short lengths of mesh coated with the various surfactants weresandwiched between a polyvinyl chloride (PVC) sheet overprinted with asilver/silver chloride ink and a transparent polyester film by means ofdouble-sided adhesive tape. This arrangement provided a model samplechamber having a height of approximately 180 μm, as measured by amicrometer. The use of a hydrophobic silver/silver chloride layer on thesurface of one side of the model sample chamber was intended toreproduce more closely the environment within a typical commerciallyavailable sample chamber.

Freshly drawn venous blood (10 μl) was applied by automatic pipette(Gilson) to the edge of the model sample chamber. The progress of theblood as it was drawn into the sample chamber was recorded by ahigh-speed video camera at a speed of 16 frames per second. Examples ofcaptured video images are shown in FIG. 3. The small number in the upperleft corner of each frame represents the number of the frame from theintroduction of the sample. The surfactant used was G2109. No spreadingof blood in the model sample chamber was seen for the uncoated control.Blood did wick into model sample chambers containing mesh coated withsurfactants FC-170C (control), G2109, PP3, DC 193, DC 190, and FSN-100.However, the wicking speed for blood was observed to be slower than thatrecorded for water in Example 1. This result is expected because of thehigher viscosity of blood relative to that of water.

TABLE 3 Identifier Trade Name Spreading of blood seen G2109 Atlas G2109Yes DC 193 Dow Corning 193 Yes DC 190 Dow Corning 190 Yes FSN-100 ZonylFSN-100 Yes PP3 Duron PP3 Yes FC-170C Fluorad FC-170C Yes (prior artcontrol) N/A N/A No (uncoated control)

Example 3

The purpose of this example was to qualitatively assess the spreadingability of blood onto a nylon mesh coated with surfactants, where thenylon mesh is incorporated into a biosensor having two layers of mesh.

Biosensors substantially similar to those described in U.S. Pat. No.5,628,890, incorporated herein by reference, were constructed. Thebiosensors contained a working electrode, an electrode that performs asa reference electrode and a counter electrode, and a trigger electrode.The sample chamber of the biosensor contained two layers of nylon mesh,both of which were coated with surfactant. The layers of nylon mesh weredesignated NY64 and NY151, both supplied by Sefar (Switzerland). Adip-coating technique was used to coat separate rolls of each type ofmesh with the various surfactants being evaluated. TABLE 4 shows theidentity and amount of surfactant coated on each layer of mesh.

Freshly drawn venous blood (5 μl) was applied by automatic pipette(Gilson) to the edge of the sample chamber. The progress of the blooddrawn into the sample chamber was recorded by a high-speed video cameraat a speed of 16 frames per second. Examples of captured video imagesare shown in FIG. 4. Blood wicked into the sample chambers of thebiosensors containing mesh coated with surfactants FC-170C (control),G2109, DC 193, DC 190, and FSN-100.

TABLE 4 Concentra- Concentration tion of of surfactant surfactant (%w/w) (% w/w) Spreading in NY64 in NY151 of Identifier Trade Name coatingbath coating bath blood seen G2109 Atlas G2109 1% 1% Yes DC 193 DowCorning 193 1% 1% Yes DC 190 Dow Corning 190 1% 1% Yes FSN-100 ZonylFSN-100 1% 1% Yes FC-170C Fluorad FC-170C 3.5%   0.35%   Yes (prior artcontrol)

Example 4

The purpose of this example was to qualitatively assess the spreadingability of blood onto a nylon mesh coated with surfactants, where thenylon mesh is incorporated into a biosensor having two layers of mesh.The biosensors were stored 18 months prior to testing.

Biosensors of the type described in Example 3 were sealed in foilpackets and stored at ambient temperature for 18 months. The biosensorswere then tested with blood, and the average time of filling for thesample chambers were determined from recorded video sequences (TABLE 5).

In each run, a sample of freshly drawn venous blood was applied byautomatic pipette (Gilson) to the edge of the sample chamber. Thesamples of blood were drawn from two donors. Each sample contained 10 μlof blood. For each type of surfactant, six biosensors were tested withblood samples from one donor and six biosensors were tested with bloodsamples from the other donor. The progress of the blood as it was drawninto the sample chamber was recorded by a high-speed video camera at aspeed of 16 frames per second.

Sample chambers containing mesh coated with the surfactant G2109 werenot stable and failed to fill with blood. The best performance withrespect to filling was observed for the sample chambers containing meshcoated with surfactant DC 193. The filling speed for the sample chamberscontaining mesh coated with the surfactant DC 193 exceeded that of thesample chambers containing mesh coated with the surfactant FC-170C.Sample chambers utilizing mesh coated with the surfactant FSN-100required almost twice as much time to fill as did sample chambersutilizing mesh coated with surfactant DC 193. Sample chambers containingmesh coated with the silicone surfactant DC 190 displayed fillingperformance comparable to that of sample chambers containing mesh coatedwith the surfactant FC-170C.

TABLE 5 Concentration Concentra- of tion of surfactant surfactant (%w/w) in Average (% w/w) NY151 time to in NY64 coating fill sampleIdentifier Trade Name coating bath bath chamber G2109 Atlas G2109 1% 1%Failed to fill DC 193 Dow Corning 193 1% 1% 2.7 sec DC 190 Dow Corning190 1% 1% 3.4 sec FSN-100 Zonyl FSN-100 1% 1% 5.1 sec FC-170C FluoradFC-170C 3.5%   0.35%   3.2 sec (prior art control)

Example 5

The purpose of this example was to qualitatively assess the spreadingability of blood onto a polyester mesh, where the polyester mesh isincorporated into a biosensor having one layer of mesh. The biosensorswere stored 18 months prior to testing.

Biosensors substantially similar to that described in WO 99/19507,published 22 Apr. 1999, incorporated herein by reference, wereconstructed, with the exception that the sample chamber of the biosensorcontained a single layer of polyester mesh coated with a surfactant. Thebiosensors contained a working electrode, an electrode that performs asa reference electrode and a counter electrode, and a trigger electrode.The sample was introduced at the end of the biosensor strip, not throughan aperture in the cover layer. The layer of polyester mesh was PE130mesh supplied by Sefar (Switzerland). A dip coating method was used tocoat separate rolls of PE130 mesh with the various surfactants underevaluation (TABLE 6). Aqueous isopropanol solutions of surfactants wereused for coating.

In each run, a sample of freshly drawn venous blood was applied byautomatic pipette (Gilson) to the edge of the sample chamber. Thesamples of blood were drawn from two donors. Each sample contained 10 μlof blood. For each type of surfactant, six biosensors were tested withblood samples from one donor and six biosensors were tested with bloodsamples from the other donor. The progress of the blood as it was drawninto the sample chamber was recorded by a high-speed video camera at aspeed of 16 frames per second.

Biosensors containing the surfactant G2109 were not stable and failed tofill. The best performance with respect to filling was observed for thesample chambers containing mesh coated with the surfactant DC 193. Thefilling speed for sample chambers containing mesh coated with thesurfactant DC 193 exceeded that of the sample chambers containing meshcoated with the surfactant FC-170C. The sample chambers containing meshcoated with the surfactant DC 190 exhibited greater filling speed ascompared with sample chambers containing mesh coated with the surfactantFC-170C, but were deemed unsuitable on the ground of very poor precisionof electrode response. The surfactant DC 190 contains both EO and POwhereas the surfactant DC 193 contains only EO (TABLE 1).

TABLE 6 Concentration of surfactant (% w/w) in Average time to PE130coating fill sample Identifier Trade Name bath chamber G2109 Atlas G21091% Failed to fill DC 193 Dow Corning 193 1% 1.9 sec DC 190 Dow Corning190 1% 2.4 sec FSN-100 Zonyl FSN-100 1% 2.6 sec FC-170C Fluorad FC-170C3% 2.9 sec (prior art control)

Example 6

The purpose of this example was to quantitatively determine, by infrared(IR) spectroscopy, the quantity of silicone surfactant DC 193 coatedonto nylon and polyester mesh. The stability of a biosensor is dependentupon the quantity of surfactant applied to the layer of mesh.

Various solutions of the surfactant DC 193 in water with approximately5% isopropylalcohol (depending on concentration of the surfactant DC193) were used as coating solutions. Polyester mesh (PE130, roll widthof approximately 1 m) was passed through the coating solution anddirected between two pinching rollers at a constant speed. The mesh wasthen dried at a temperature of 120° C. After the side edges of the rollof mesh were removed, the dried mesh was slit into rolls having a widthof 14 mm. The coating process was performed by Sefar (Switzerland).

A length of polyester mesh (10 cm, PE130) was taken from a roll (widthof 14 mm) and cut into two five (5) cm lengths. Each sample was weighedand then placed in a sealed glass test tube. Cyclohexane (5 ml; minimumpurity 99.8%) was added to the test tube, and the test tube shaken for15 minutes on an orbital shaker at a speed of 1400 rpm. The resultingsolution was tested by FT-IR using a liquid sample cell having 1 mm pathlength having barium fluoride windows. The scan conditions were: 4scans, 4 cm⁻¹ resolution, 1200 to 1050 cm⁻¹ range. The total peak areawas calculated for the absorbance spectrum from 1145 to 1080 cm⁻¹. Acalibration curve, obtained by measurement of known concentrations ofthe surfactant DC 193 in cyclohexane, was used to determine the quantityof the surfactant DC 193 in the extracted solution, and, consequently,the coating weight of the surfactant on the mesh sample.

The method described above can be used in a similar manner to determinethe coating weight of the surfactant DC 193 on the nylon meshes NY64 andN151.

A linear relationship was observed between the concentration ofsurfactant used in the coating solution and the weight of the surfactantcoated on the polyester mesh PE130 (see FIG. 5) and on the nylon meshesNY64 (see FIG. 6) and NY151 (see FIG. 7), as determined by FT-IR andexpressed in terms of μg of surfactant per mg of mesh.

Example 7

The purpose of this example was to determine the frequency of bloodfilling of biosensors having a single layer of mesh containing thesurfactant DC 193 coated thereon, as a function of concentration ofsurfactant, storage temperature, and storage time.

Biosensors substantially similar to that described in Example 5 wereused, except that the sample was introduced through an aperture punchedthrough the biosensor. Various coating weights were used. The layer ofpolyester mesh was PE130 mesh, supplied by Sefar (Switzerland). Separaterolls of PE130 mesh were dip coated in aqueous isopropanol solutions ofthe surfactant DC 193.

The biosensors were packaged in foil and stored at ambient temperature(22° C.), 30° C., 40° C., and 50° C., for the purposes of the example.Ninety-six biosensors at each concentration of the surfactant DC 193 andat each storage temperature were tested at regular intervals todetermine the percentage of the biosensors that could be filled withblood. In each run, a sample of freshly drawn venous blood was appliedby automatic pipette (Gilson) to the edge of the sample chamber. Thesamples of blood were drawn from two donors. Each sample contained 10 μlof blood. Forty-eight biosensors were tested with blood samples from onedonor and forty-eight biosensors were tested with blood samples from theother donor.

The stability profiles of the biosensors were determined by plotting thepercentage of the sample chambers of the biosensors that filled againstthe square root of the storage period (see FIG. 8A). Initially, 100% ofthe sample chambers filled with blood, but as storage time increased,this percentage declined until it was not possible to fill any samplechambers with blood. The stability profile can be enhanced by increasingthe coating concentration of the surfactant DC 193 and decreasing thestorage temperature. This enhancement is clearly seen in FIG. 8B. Theshelf life required for the biosensor determines the minimum amount ofthe surfactant DC 193 that must be coated onto the layer of mesh.Biosensors containing polyester mesh coated with the surfactant FC-170Cexhibited significantly more stability than did those biosensors havingpolyester mesh coated with the surfactant DC 193. For example, nofailure of biosensors having mesh coated with FC-170C surfactant wasobserved at the storage temperature of 50° C. even after one year ofstorage. However, such stability is considerably in excess of thatnormally required (12 to 24 months at a storage temperature of 30° C.),and this requirement is achieved by coating the layer of mesh with thesurfactant DC 193 at a sufficiently high concentration. Shelf life ofthe biosensor for a requirement of 100% filling at a storage temperatureof 30° C. can be predicted from a plot such as that shown in FIG. 9.

Example 8

The purpose of this example was to determine the time of filling and thefrequency of filling of biosensors having two layers of mesh in thesample chamber as a function of the concentration of surfactant, storagetemperature, and storage time.

Biosensors of the type described in Example 3 were used. The samplechambers of the biosensors contained two layers of nylon mesh, bothcoated with the surfactant. The layers of nylon mesh were NY64 mesh andNY151 mesh, both supplied by Sefar (Switzerland). Separate rolls of eachtype of mesh were dip coated in a solution containing the surfactant DC193. The concentration of the surfactant in the coating bath for theNY151 mesh was 0.09% DC 193. For the NY64 mesh, the concentrations ofthe surfactant in the coating bath were 0.35 and 1.05%.

The biosensors were packaged in foil and stored at ambient temperature(22° C.), 30° C., 40° C., and 50° C. for the purposes of the example.Ninety-six biosensors at each concentration of DC 193 at each storagetemperature were tested at regular intervals to determine the percentagethat can be filled with blood. In each run, a sample of freshly drawnvenous blood was applied by automatic pipette (Gilson) to the edge ofthe sample chamber. The samples of blood were drawn from two donors.Each sample contained 10 μl of blood. Forty-eight biosensors were testedwith blood samples from one donor and forty-eight biosensors were testedwith blood samples from the other donor.

The stability profiles of the biosensors were assessed by plotting thepercentage of the sample chambers that filled and the time of filling ofthe sample chambers as a function of the square root of storage time(see FIGS. 10A, 10B, 11A, 11B). Initially, 100% of the sample chambersfilled with blood, but as the storage time increased, this percentagedeclined. Testing was terminated at 52 weeks. Time of filling wasobserved to increase as a function of the storage time and even more soas the storage temperature increased. The stability of filling wasenhanced by increasing the coating concentration of the surfactant DC193 and decreasing the storage temperature. Approximately 100% of thesample chambers of the biosensors filled in under five (5) seconds whenstored at a temperature of 30° C. for a minimum of 12 months.

Example 9

The purpose of this example was to determine the time of filling of abiosensor having a single layer of mesh in the sample chamber as afunction of concentration of surfactant and storage time at 50° C., witha variety of silicone surfactants coated onto the layer of mesh.

Biosensors similar to that described in Example 5 were used. The layerof polyester mesh was PE130 mesh supplied by Sefar (Switzerland). A dipcoating method was used to coat separate rolls of PE130 mesh with thevarious surfactants being evaluated (TABLE 7). Aqueous isopropanolsolutions of surfactants at concentrations of 1% w/w and 3% w/w wereused for coating. A 50:50 mixture of the surfactants BC2213 and BC2234was also evaluated.

The biosensors were packaged in foil and stored at a temperature of 50°C. for the purposes of the example. A sample of freshly drawn venousblood was applied by automatic pipette (Gilson) to the edge of thesample chamber. The samples of blood were drawn from two donors. Eachsample contained 5 μl of blood. For each type of surfactant, sixbiosensors were tested with blood samples from one donor and sixbiosensors were tested with blood samples from the other donor. Theprogress of the blood as it was drawn into the sample chamber wasrecorded by a high-speed video camera at a speed of 16 frames persecond.

TABLE 7 Trade Type of surfactant and Identifier name Manufacturerapproximate structure DC 193 Dow Dow Corning Silicone surfactantcontaining Corning ethylene oxide (EO) 193 MD₉(D′E₁₂H)₄M DC 190 Dow DowCorning Silicone surfactant containing Corning ethylene oxide (EO) and190 propylene oxide (PO) MD₁₀(D′E₁₈P₁₈H)₄M FC-170C Fluorad 3MPerfluoroalkylsulfonamido FC-170C oxyethylene adduct (prior artC₈F₁₇SO₂N(Et)(CH₂CH₂O)_(n)—H control) BC2213 BC2213 Basildon Trisiloxanesurfactant containing Chemicals ethylene oxide (EO) M(D′E₁₂H)₄M BC2234BC2234 Basildon Silicone surfactant containing Chemicals ethylene oxide(EO), methyl- capped MD₉(D′E₁₂Me)₄MThe standard nomenclature for silicone surfactants is used, where:

M represents (CH₃)₃SiO

D represents (CH₃)₂SiO

D′ represents (CH₃)SiO substituted with an EO or EO/PO polymeric chainlinked with a C₃H₆ group. The polymer chain is terminated with a groupR, which can be hydrogen, alkyl (preferably methyl), ester (preferablyacetate)

E represents ethylene oxide (EO)

P represents propylene oxide (PO)

For example:MD_(x)(D′E_(y)R)_(z)M represents(CH₃)₃SiO—((CH₃)₂SiO)_(x)—[CH₃SiO—{C₃H₆—(C₂H₄O)_(y)—R}]_(z)—OSi(CH₃)₃The average time of filling was calculated for biosensors containingdifferent types of surfactants as a function of storage time (0-12weeks) at a temperature of 50° C. The resulting data is shown in FIG.12.

Stability failure points for the biosensors are indicated by largeincreases in time of filling. Stability failure was observed for 1%BC2213, 3% BC2213 and 1% BC2213 (50%)/BC2234 (50%). Increased stabilitycan be brought about by increasing the weight of surfactant on the meshlayer (compare 3% BC2213 versus 1% BC2213). The stability of thesilicone surfactants follows the approximate order from the greatest tothe lowest:[FC-170C (control)]>DC 193>C2234>BC2234 (50%)/BC2213 (50%)>BC2213The low molecular weight trisiloxane surfactant BC2213 is the leaststable, but a mixture of BC2213 with BC2234 exhibits increasedstability.TABLE 8 compares initial time of filling for biosensors containing thepolyester mesh coated with various silicone surfactants with the initialtime of filling of biosensors containing the polyester mesh coated withsurfactant FC-170C. The time of filling follows the approximate orderfrom lowest to greatest:BC2213>BC2234˜BC2234 (50%)/BC2213 (50%)>DC 193>FC-170C (control)The time of filling for all the sample chambers containing mesh coatedwith silicone surfactants are lower than those for sample chamberscontaining the mesh coated with the surfactant FC-170C (control). Thetrend for time of filling is the converse of that seen for fillingstability. Mixtures of silicone surfactants can provide the bestcompromise of filling speed and stability, e.g., a mixture of thesurfactants BC2213 (rapid filling and poor stability) and BC2234 or DC193 (slow filling and good stability).

TABLE 8 Concentration of Average time to fill coating bath for samplechamber at t = 0 Identifier PE130 (% w/w) weeks DC 193 1% 1.60 sec DC193 3% 1.93 sec FC-170C 3% 2.25 sec BC2213 1% 1.03 sec BC2213 3% 1.17sec BC2234 1% 1.18 sec BC2234 3% 1.07 sec BC2213 1% 1.22 sec(50%)/BC2234 (50%) BC2213 3% 1.12 sec (50%)/BC2234 (50%)

Example 10

The purpose of this example was to qualitatively assess the adhesion ofa polyester film and an insulating ink to a layer of nylon mesh coatedwith various surfactants in a biosensor having two layers of mesh.

Biosensors of the type described in Example 3 were used. The samplechambers of the biosensors contained two layers of nylon mesh, bothcoated with a surfactant. The layers of nylon mesh were NY64 and NY151,supplied by Sefar (Switzerland). The surfactants (G2109, DC 190, DC 193,FSN-100 and FC-170C), listed in TABLE 4, were included in this example,along with the anionic surfactant Aerosol OT-100 (listed in TABLE 1).The biosensors were assessed for any compatibility or adhesion problemsor both between the various surfactants and any other materials.

In U.S. Pat. No. 5,628,890, the surfactant-coated mesh layer was held inplace by overprinting with a layer of insulating ink (Sericard,commercially available from Sericol, Broadstairs, UK). The surfactantslisted in TABLE 4 (G2109, DC 190, DC 193, FSN-100 and FC-170C) werecompatible with the insulating ink. The polar anionic surfactant OT-100was incompatible with the organic ink, with the result that it was notpossible to adhere mesh coated with that surfactant to the surface ofthe electrode. For this reason, the surfactant OT-100 was rejected andno further work was carried out with it.

The surfactant-coated mesh layer also came into contact with a top layerof polyester film in those biosensors constructed according to U.S. Pat.No. 5,628,890. It is necessary to provide good adhesion between thepolyester film and the surfactant-coated mesh where the sides of thesample chamber of the biosensor are formed. If good adhesion is notprovided, the blood in the sample chamber may seep between the polyesterfilm and the mesh that is being held in place by the insulating ink atthe edge of the biosensor. The consequences of such seepage are that (1)the volume of blood required by the biosensor will be increased, and (2)the blood is not contained within the sample chamber, which may causetrouble with handling. The surfactants listed in TABLE 4 (G2109, DC 190,DC 193, and FC-170C), with the exception of FSN-100 provided biosensorswherein the polyester film adhered sufficiently to the insulating layerwith little or no seepage of blood. The surfactant FSN-100 yieldedbiosensors having poorly adhering polyester film, as demonstrated inFIGS. 13A and 13B. The surfactant DC 193 performed well in this respect.

Various modifications and alterations of this invention will becomeapparent to those skilled in the art without departing from the scopeand spirit of this invention, and it should be understood that thisinvention is not to be unduly limited to the illustrative embodimentsset forth herein.

1. A method for determining the concentration of an analyte in a sample,the method comprising: applying the sample to a biosensor, the biosensorcomprising: a substrate; a working electrode; a counter electrode; acover layer defining an enclosed space over the electrodes; and at leastone mesh layer disposed in the enclosed space between the cover layerand the electrodes, the at least one mesh layer coated with at least onesilicone surfactant, wherein the at least one silicone surfactantcomprises a mixture of silicone surfactants; applying a voltage to theworking electrode; measuring a current at the working electrode; andcorrelating the current measured with the concentration of the analytein the sample.
 2. The method according to claim 1, wherein the mixtureof silicone surfactants comprises a high molecular weight siliconesurfactant and a low molecular weight silicone surfactant.
 3. The methodof claim 2, wherein the high molecular weight silicone surfactant andthe low molecular weight silicone surfactant have the formula:

wherein x ranges from 8 to 9, inclusive, y ranges from 3 to 4,inclusive, z ranges from 11 to 13, inclusive, and R being hydrogen forthe high molecular weight silicone surfactant; and x is 0, y is 1, zranges from 3 to 15, inclusive, and R is hydrogen, methyl, or acetatefor the low molecular weight silicone surfactant.
 4. The method of claim2, wherein the weight fraction of the high molecular weight siliconesurfactant in the mixture ranges from 1% to 99%, inclusive.
 5. Themethod of claim 2, wherein the weight fraction of the low molecularweight silicone surfactant in the mixture ranges from 1% to 99%inclusive.
 6. The method of claim 1, wherein the working electrodecomprises a working ink, the working ink comprising an analyteresponsive enzyme and a mediator.
 7. The method of claim 6, wherein theanalyte responsive enzyme is selected from the group consisting ofglucose oxidase, glucose dehydrogenase and 3-hydroxybutyratedehydrogenase.
 8. The method of claim 1, wherein the biosensor comprisesa single layer of mesh.
 9. The method of claim 1, wherein the biosensorcomprises two layers of mesh.
 10. The method of claim 1, wherein the atleast one mesh layer comprises polyester or polyamide.
 11. The method ofclaim 1, wherein the at least one mesh layer is less than 150 microns inthickness.
 12. The method of claim 1, wherein the at least one meshlayer comprises fibers having a diameter from about 70 to about 100microns.
 13. A method for determining the concentration of an analyte ina sample, the method comprising: applying the sample to a biosensor, thebiosensor comprising: a substrate; a working electrode; a counterelectrode; a cover layer defining an enclosed space over the electrodes;and at least one mesh layer disposed in the enclosed space between thecover layer and the electrodes, the at least one mesh layer coated withat least one silicone surfactant, wherein the at least one siliconesurfactant comprises a trisiloxane surfactant; applying a voltage to theworking electrode; measuring a current at the working electrode; andcorrelating the current measured with the concentration of the analytein the sample.
 14. The method of claim 13, wherein the working electrodecomprises a working ink, the working ink comprising an analyteresponsive enzyme and a mediator.
 15. The method of claim 14, whereinthe analyte responsive enzyme is selected from the group consisting ofglucose oxidase, glucose dehydrogenase and 3-hydroxybutyratedehydrogenase.
 16. The method of claim 13, wherein the biosensorcomprises two layers of mesh.
 17. The method of claim 13, wherein the atleast one mesh layer comprises polyester or polyamide.
 18. The method ofclaim 13, wherein the at least one mesh layer is less than 150 micronsin thickness.
 19. The method of claim 13, wherein the at least one meshlayer comprises fibers having a diameter from about 70 to about 100microns.
 20. A method for determining the concentration of an analyte ina sample, the method comprising: applying the sample to a biosensor, thebiosensor comprising: a substrate; a working electrode; a counterelectrode; a cover layer defining an enclosed space over the electrodes;and at least one mesh layer disposed in the enclosed space between thecover layer and the electrodes, the at least one mesh layer coated withat least one silicone surfactant, wherein the at least one siliconesurfactant comprises a cyclosiloxane surfactant; applying a voltage tothe working electrode; measuring a current at the working electrode; andcorrelating the current measured with the concentration of the analytein the sample.
 21. The method of claim 20, wherein the working electrodecomprises a working ink, the working ink comprising an analyteresponsive enzyme and a mediator.
 22. The method of claim 21, whereinthe analyte responsive enzyme is selected from the group consisting ofglucose oxidase, glucose dehydrogenase and 3-hydroxybutyratedehydrogenase.
 23. The method of claim 20, wherein the biosensorcomprises two layers of mesh.
 24. The method of claim 20, wherein the atleast one mesh layer comprises polyester or polyamide.
 25. The method ofclaim 20, wherein the at least one mesh layer is less than 150 micronsin thickness.
 26. The method of claim 20, wherein the at least one meshlayer comprises fibers having a diameter from about 70 to about 100microns.